Lyophilized cyclophosphamide

ABSTRACT

A lyophilized pharmaceutical solid composition containing cyclophosphamide for reconstitution with water to provide a solution for oral or parenteral administration. This lyophilized cyclophosphamide solid composition demonstrates improved stability, solubility characteristics and enhanced appearance compared with currently available dry powder pre-mix compositions of cyclophosphamide. The lyophilized solid composition contains about 20 parts by weight of cyclophosphamide, about 11/4-2 parts by weight of water and from about 10-85 parts by weight of excipient which is comprised mainly of mannitol. Processes for making the composition are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This is a continuation of Ser. No. 589,202, which was filed Mar. 13, 1984 and is now issued as U.S. Pat. No. 4,537,883 and was itself a continuation-in-part of Ser. No. 440,906, filed Nov. 12, 1982, and now abandoned.

BACKGROUND OF THE INVENTION

Cyclophosphamide is the generic name for 2-[bis(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide monohydrate, a widely used antineoplastic drug chemically related to the nitrogen mustards. Cyclophosphamide was one example of a group of novel cyclic phosphoric acid ester amides which were disclosed and claimed in U.S. Pat. No. 3,018,302 granted Jan. 23, 1962 to H. Arnold, et al.

A related series of compounds bearing substituents on the oxazaphosphorine ring nitrogen was disclosed and claimed in U.S. Pat. No. 3,732,340 granted May 8, 1973 also to H. Arnold, et al.

Over the past 20 years a considerable amount of literature concerning cyclophosphamide has accumulated. Most of these references deal mainly with clinical applications of this agent as an antineoplastic drug. Cyclophosphamide has been distributed for much of that time by Mead Johnson & Company under the proprietary name CYTOXAN®. ENDOXAN® and NEOSAR® are proprietary names for similar pharmaceutical formulations of cyclophosphamide which are essentially comparable to CYTOXAN®.

While cyclophosphamide comprises the monohydrate drug form, which is the easiest to isolate and with which to work, the anhydrous form also exists. No other hydrate form has been reported. As used herein, the term "cyclophosphamide" refers generically to the drug substance (i.e., either the monohydrate or the anhydrous form) the term "cyclophosphamide monohydrate" refers specifically to the monohydrate and the term "anhydrous cyclophosphamide" refers to the anhydrous form. The monohydrate form is preferred for pharmaceutical processing, as the anhydrous form readily picks up water to form the monohydrate when exposed to a relative humidity of about 20-30% or higher at about 25° C. While the monohydrate is stable, nonetheless, under dry conditions (relative humidities of about 20% or less) the monohydrate begins to lose this water of hydration which can cause problems in manufacture. Because of stability limitations which may be due in part to ready interconversion between the anhydrous and monohydrate forms, it is recommended that storage temperatures for cyclophosphamide products not exceed 30° C. (86° F.), and preferably be stored at about 25° C. (77° F.).

Currently, the parenteral dosage formulations of cyclophosphamide consist of sterile packaged dry powder blend admixtures of cyclophosphamide monohydrate and sodium chloride. These premixes are dissolved in water prior to administration which can be oral as well as parenteral. It is intended that the solution itself be administered promptly after being prepared but it is satisfactory for use up to several hours after preparation. During processing and/or storage of the present dry powder premix formulation, a glassiness and/or stickiness can be acquired by the premix composition giving an unattractive material with inferior solubility characteristics and decreased potency. This deterioration is more pronounced as storage time is extended or if the upper limit of the storage temperature range is exceeded.

A common practice used with constitution of sterile solids by a suitable aqueous vehicle consists of warming the container to expedite the dissolution process, especially when the solids dissolve slowly. A study examining the effect of briefly heating cyclophosphamide solutions was reported by D. Brooke, et al. in American Journal of Hospital Pharmacy, 32:44-45 (1975). This study concluded that warming vials of cyclophosphamide in order to facilitate dissolution after adding an aqueous vehicle could decrease the potency of the final injectable product. In summary, these stability limitations and dissolution difficulties can often result in clinical usage of subpotent cyclophosphamide solutions.

Therefore, the main objective of the project culminating in the instant invention was to provide a cyclophosphamide dosage form with improved solubility characteristics and enhanced appearance, while maintaining stability comparable to the dry pre-mix composition. Unexpectedly, the unique lyophilized solid composition discovered has improved stability over the previous dry pre-mix composition.

Problems with unstable drug solutions have been handled previously by practitioners in the pharmaceutical arts by applying lyophilization, cf: L. Lachman, et al, "The Theory and Practice of Industrial Pharmacy", 2nd Ed., Lea & Febiger, Philadelphia, Pa., pp. 521-524 (1976); "Remington's Pharmaceutical Sciences", 15th Ed., Mack Publishing Co., Easton, Pa., pp. 1483-1485 (1975). The technique known as lyophilization is often employed for injectable pharmaceuticals which exhibit poor stability in aqueous solution. This process involves freeze drying, whereby ice is sublimed from frozen solutions leaving only the solid, dried components of the original liquid.

The particular advantages of lyophilization are that biologicals and pharmaceuticals which are unstable in aqueous solution yet relatively stable in the solid state can be processed and filled into dosage containers in solution, taking advantage of the relative ease of processing a liquid; dried without elevated temperatures, thereby eliminating adverse thermal effects; and then stored in the dry state in which there are relatively few stability problems. Additionally, freeze dried products are often more soluble and/or more rapidly solubilized, dispersions are stabilized, and products subject to degradation by oxidation or hydrolysis are protected.

Pharmaceuticals to be freeze dried are usually in aqueous solution ranging from 0.01 to 40% in concentration of total solids. Final moisture content of the dried product is generally below 1.0% although some products, mainly biologicals, may have a final moisture content which could range as high as about 10%. Usually, the improvement in stability of the lyophilizate, compared to the solution, is due to the absence of water in the pharmaceutical composition.

The active constituent of many pharmaceutical products, though, is present in such small quantity that if freeze-dried alone, it may not give a composition of suitable bulk and, in some cases, its presence would be hard to detect visually. Therefore, excipients are often added to increase the amount of solids present. In most applications it is desirable for the dried product cake to occupy essentially the same volume as that of the original solution. To achieve this, the total solids content of the original solution is usually about 10 to 25%. Among substances found useful for this purpose, often in combination, are sodium or potassium phosphates, citric acid, tartaric acid, gelatin, lactose and other carbohydrates such as dextrose, mannitol, and dextran; and on occasion, preservatives. Various excipients contribute appearance characteristics to the cake, such as whether dull and spongy or sparkling and crystalline, firm or friable, expanded or shrunken, and uniform or striated. Therefore, formulation of a composition to be freeze dried must include consideration not only of the nature and stability characteristics required during the liquid state, both freshly prepared and when reconstituted before use, but the characteristics desired in the final lyophilized cake. Additionally, for products to be reconstituted for parenteral usage, consideration must also be given to the pharmacological effects of excipients chosen. In some instances, there may even be chemical interaction between the active ingredient and one or more of the excipients during processing. This could, of course, result in reduced potency of the finished product.

For all the above reasons, it becomes apparent that selection of a suitable excipient or excipients for a pharmaceutical product containing a reactive, thermally labile, and inherently unstable ingredient such as cyclophosphamide is not an obvious process. Considerable testing, including drug assay, would be required, as in the instant case, for the development of such a composition. We surprisingly found that only the use of mannitol as the primary excipient gave a far superior lyophilizate compared to lyophilizates obtained using other excipients. Unexpectedly, we also discovered that a lyophilized cyclophosphamide solid composition containing about 4% moisture gave a product with superior thermal stability, compared to currently available dry powder premixes, lyophilized cyclophosphamide solid compositions with moisture levels of about 1% or less, or even cyclophosphamide itself.

Summary of the Invention

This invention concerns an improved solid pharmaceutical composition and is based in part upon the discovery that a lyophilized cyclophosphamide-mannitol solid composition has improved thermal stability when it contains an amount of water approximately equimolar to the cyclophosphamide content taken as the anhydride. Of equal importance is the discovery that the desirable physical properties of the solid composition appear to be achieved only by using mannitol as the major excipient. Two processes have been developed which allow facile production of the solid composition in vials for packaging as unit dosage forms for reconstitution as sterile solutions.

DETAILED DESCRIPTION OF THE INVENTION

The therapeutically-active component of this invention, cyclophosphamide, is a well known and widely used anticancer agent. Cyclophosphamide chemically is 2-[bis-(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine-2-oxide monohydrate, shown in Formula I ##STR1## It is appreciated by the practitioner that the degree of reactivity desired for efficacy in an agent of this sort necessarily acts as a limit to its inherent stability in aqueous solution. Mainly for this reason, cyclophosphamide is compounded as a sterile dry powder mixture of cyclophosphamide monohydrate and NaCl for constitution with sterile water just prior to administration. Some instability of cyclophosphamide dry powder mixes can be evidenced by change in physical properties, e.g. decreased rate of dissolution, change in appearance, and the like, as well as loss of potency on storage. This deterioration becomes more pronounced on extended storage or on exposure of the powder mix to temperatures above about 90° F., a temperature not uncommon during commercial transportation or warehouse storage. Freshly prepared solutions made up from this powder mix can exhibit lowered potency due not only to prior degradation of the active ingredient but also through incomplete dissolution of the powder mix as well.

The instant invention has resulted from work undertaken to ascertain if physical properties, especially dissolution rate, could be improved in a lyophilized composition containing anhydrous cyclophosphamide.

Cyclophosphamide was thoroughly tested with those excipients compatible with parenteral administration to determine if a suitable lyophilizate cake could be formed by freeze-drying. Following the lyophilization process, the resulting cake was evaluated visually on its physical appearance using as desired criteria: original shape, no shrinkage or meltback, good coloration, homogeneity, firmness, and crystallinity. The dissolution rate was then tested and a global score of poor, fair, or good, based on the above criteria, was assigned, cf: Table 1.

                  TABLE 1                                                          ______________________________________                                         Excipient Testing For Cake Formation                                                        Pre-lyophilization                                                             Solution:                                                                      Concentration Lyophilization                                      Excipient    (% W/V)       Result                                              ______________________________________                                         Lactose      1.0, 2.0, 2.5, 3.3, 3.5,                                                                     Poor cakes                                                       5.0, 6.7, 8.0                                                     Dextrose     2.0, 2.5      Poor cakes                                          Tartaric acid                                                                               2.0           Poor cake                                           Urea         1.0, 2.0, 2.5 Poor cakes                                          L-arginine   0.5           Poor cake                                           Polyvinylpyrrolidone                                                                        3.0           Poor cake                                           (PVP)                                                                          KH.sub.2 PO.sub.4                                                                           1.0           Poor cake                                           K.sub.2 HPO.sub.4                                                                           1.0           Poor cake                                           Na.sub.2 CO.sub.3                                                                           1.0           Poor cake                                           NaHCO.sub.3  1.0, 1.25, 1.5                                                                               Poor to fair cakes                                  NaHCO.sub.3  1.75, 2.0, 2.5                                                                               Good cakes                                          Mannitol     1.7, 2.0, 2.5, 3.0, 4.0,                                                                     Good cakes                                                       5.0, 6.0, 7.0                                                     Mannitol     10.0, 15.0    Poor cakes                                          CH.sub.3 CO.sub.2 K                                                                         2.5, 3.0, 5.0 Poor cakes                                          ______________________________________                                    

Each solution for lyophilization contained 3.57% (W/V) cyclophosphamide monohydrate and an excipient or excipients. The solutions were prepared using Water for Injection and were filtered through a prefilter and a 0.22 μm filter membrane. The solutions were filled into 30 mL serum vials with a fill volume of 15 mL of solution per vial (the equivalent of 500 mg of anhydrous cyclophosphamide).

Prior to lyophilization, the filled vials were prechilled to 5° C. in a refrigerator. The vials were then frozen in the lyophilization chamber for approximately 2 hrs to a temperature of about -35° C. After cooling the condenser to about -60° C., the lyophilizer chamber was evacuated to approximately 50 millitorr. An initial shelf temperature of 0° C. was maintained for the first 14-16 hrs of the lyophilization cycle. The shelf temperature was then raised to 25° C. The cycle was completed when the product temperature stabilized near the shelf temperature, usually requiring 2-6 more hours, and the vacuum pressure reached equilibrium at a reading of approximately 100 millitorr. During lyophilization, product temperature was monitored using thermocouple probes placed inside several vials.

After the lyophilization process was completed, the material remaining in the vial was observed for color, appearance, texture, friability, and shrinkage from the original frozen volume. Also, each formulation was tested for its moisture loss on drying (4 hrs at 70° C. under vacuum) and its dissolution characteristics upon reconstitution with 25 mL of Sterile Water for Injection.

Unexpectedly, only two excipients from this group (Table 1) exhibited favorable cake-forming ability in the presence of cyclophosphamide. These two prototype formulations (containing NaHCO₃ or mannitol) retained their original shape, possessed suitable texture and appearance, and dissolved easily upon reconstitution with water. Most of the other excipient formulations were unsuitable due to poor quality cakes due mainly to shrinkage and/or poor dissolution. As can be seen from Table 1, only mannitol, at concentrations of 7% W/V or below, and sodium bicarbonate were initially found to be acceptable excipients in the cyclophosphamide lyophilization process. The formulations containing these two excipients dissolved easily within a few seconds upon reconstitution with water.

Further experimentation utilizing the same experimental procedure to investigate lyophilization results using mixed excipients demonstrated the surprising finding that fair to good lyophilizate cakes could be obtained only with mannitol compared to lactose or sodium bicarbonate as a primary excipient; cf: Table 2.

                                      TABLE 2                                      __________________________________________________________________________     Combined Excipient Testing                                                     Primary Excipient                                                              (Concentration                                                                           Secondary Excipient                                                  % W/V)    (Concentration % W/V)                                                                       Lyophilization Result                                   __________________________________________________________________________     Lactose (2.0)                                                                            Mannitol (1.0, 1.5)                                                                         No cakes                                                Lactose (3.3)                                                                            Citric acid, anhyd. (1.0)                                                                   No cake                                                           Na.sub.2 CO.sub.3 (1.0)                                                                     No cake                                                           NaHCO.sub.3 (1.25)                                                                          No cake                                                 NaHCO.sub.3 (1.5)                                                                        NaCl (0.25)  Poor cake, shrinkage                                              Glycine (0.25; 0.5)                                                                         Poor cakes, shrinkage                                             K.sub.2 HPO.sub.4 (0.5)                                                                     Poor cake, shrinkage                                              Citric acid, anhyd. (0.5)                                                                   Poor cake, shrinkage                                              Lactose (0.5, 1.0, 1.5)                                                                     Poor cakes, very fragile                                NaHCO.sub.3 (2.0)                                                                        Glycine (0.5)                                                                               Poor cake, very fragile                                           Na citrate (0.5)                                                                            Poor cake, very fragile                                 Mannitol (2.0)                                                                           Na ascorbate (0.33)                                                                         Poor cake, shrinkage                                              Na acetate (0.33)                                                                           Poor cake, slow dissolution                                       Na citrate (0.33)                                                                           Poor cake, shrinkage                                              Na.sub.2 CO.sub.3 (0.5)                                                                     No cake                                                           NaHCO.sub.3 (0.5)                                                                           No cake                                                           PVP (1.0)    Fair cake, sl. shrinkage                                Mannitol (2.5)                                                                           Na citrate (0.33, 0.5, 0.67)                                                                Poor cakes, shrinkage                                             L-arginine (0.5)                                                                            Poor cake, slow dissolution                                       Glycine (0.5, 1.0)                                                                          Poor cakes, unhomogenous                                          PVP (0.5, 1.0)                                                                              Fair to good cakes                                      Mannitol (3.0)                                                                           Glycine (0.5)                                                                               Fair cake, sl. shrinkage                                          Na citrate (0.33)                                                                           Good cake                                               __________________________________________________________________________

Most of the compositions listed in Table 2 exhibited some ability to form lyophilizate cakes except those in which lactose was the primary excipient; however, most of the resulting cakes were of poor quality due to friability, shrinkage and/or poor dissolution. The lyophilizates containing various secondary excipients with sodium bicarbonate were of poorer quality than the lyophilizates with sodium bicarbonate as the only excipient. One formulation containing mannitol as the primary excipient and sodium citrate and a second formulation containing mannitol as the primary excipient and polyvinylpyrrolidone (PVP) formed acceptable lyophilizates that were firm, homogenous cakes with minimum friability and shrinkage. The two combination excipient products dissolved easily in a few seconds upon reconstitution with water. These selected lyophilized solid compositions all had low moisture levels, as measured by loss on drying, on the order of about 2% or less. While considerable improvement of the dissolution properties and physical appearance was achieved with these low moisture cyclophosphamide lyophilizates, six-week stability testing indicated that these lyophilized solid compositions were heat sensitive and, in fact, much less stable than the existing sodium chloride/cyclophosphamide monohydrate dry powder premixture.

It was unexpectedly discovered, however, that humidification of these low moisture lyophilized cyclophosphamide solid compositions ("dry" lyophilizates) could be easily and reproducibly effected by subjecting them to an air atmosphere of about 60 to 80% relative humidity at about 25° C. in a closed container for 1-2 days. Under these conditions, a state of equilibrium could be achieved in which the moisture gained on a molar basis was approximately equivalent to the anhydrous cyclophosphamide content of the lyophilizate. The improved dissolution characteristics found in the "dry" cyclophosphamide lyophilizates remained essentially unchanged upon humidification. Stability testing of the humidified solid compositions ("hydrated" lyophilizates) demonstrated that those compositions containing mainly mannitol as the excipient were unexpectedly superior, compared with the sodium bicarbonate excipient compositions in maintaining minimal dissolution times, cf: Table 3.

                  TABLE 3                                                          ______________________________________                                         Comparative Stability Testing of "Hydrated" Solid                              Lyophilizates: Mannitol as excipient vs NaHCO.sub.3 as excipient               Storage    Time       Dissolution Times (sec)                                  Temperature                                                                               (Wks)      Mannitol  NaHCO.sub.3                                    ______________________________________                                         40° 3          ˜20 >180                                                      6          ˜60 >180                                                      12         ˜60 ˜120                                     35° 3          ˜20 >180                                                      6          ˜60 >180                                                      12         ˜60 ˜120                                     25° 6          ˜60 >180                                                      12         ˜60 >180                                           ______________________________________                                    

Preferred solid compositions of the instant invention are comprised of about 20 parts by weight of cyclophosphamide, taken as the anhydrous form, about 11/4-2 parts by weight of water and from about 10-40 parts by weight of excipient which is primarily mannitol. Most preferred compositions are comprised of about 20 parts by weight of cyclophosphamide, taken as the anhydride, about 11/2 parts by weight of water and about 15 parts by weight of excipient consisting essentially of mannitol.

These solid compositions may be prepared by either of two methods. The first method, of which one embodiment as mentioned hereinabove, involves lyophilization and humidification. Common to both methods is preparation of a sterile solution for lyophilization. A typical solution is composed of about 1 part by weight excipient which is mainly mannitol, about 1.5 parts by weight cyclophosphamide (as the monohydrate), and about 40 parts water. In order to obtain optimal results with the lyophilizate composition this solution should not contain much more than about 4% cyclophosphamide content. Continuing with the first method, this solution is aseptically filled into suitable containers and then lyophilized to a "dry" lyophilizate cake which has a moisture content on the order of about 2% or less. This lyophilization is done in a short period of time (˜24 hrs) at a lyophilization chamber pressure of about 100 to 500 millitorr using a very cold condenser temperature (about -60° C.) and a warm shelf temperature (about 20°-25° C.). This typical "dry" lyophilizate is then humidified to give a "hydrated" lyophilizate containing approximately 4% moisture. With humidification at atmospheric pressure, further water pick-up essentially stops at this point giving the stable "hydrated" lyophilizate solid composition of the instant invention. The amount of water taken up is approximately equimolar ranging to about 30% excess compared with the cyclophosphamide (on an anhydrous basis) present in the cake. While the humidification can be done at approximately atmospheric pressure, the time required for complete humidification is considerably shortened (to at least a third or less) when done under reduced pressure. By reduced pressure is meant a pressure of about 0.1 to 25 torr. A unit of 1 torr is equivalent to 1/760 standard atmospheric pressure.

A second method of obtaining the desired lyophilized solid composition involves lyophilizing the sterile solution directly using a longer lyophilization cycle (˜48 hrs). This method, done more slowly under milder conditions, requires adjustment of certain parameters of the lyophilization process such as shelf temperature, and condenser temperature during the process of drying. This second lyophilization process comprises 2 stages of freeze-drying done under a 500 millitorr chamber pressure. The first stage of freeze-drying is done with a low condenser temperature of about -60°, and a shelf temperature at about 10° C. The second stage of freeze-drying requires raising the condenser temperature to about -30° C. and lowering the shelf temperature to about -10° C.

These lyophilized cyclophosphamide solid compositions may be provided in single dose container forms by aseptically filling suitable containers with the sterile pre-lyophilization solution to a prescribed cyclophosphamide content; preparing the desired lyophilized solid composition, using either of the two methods described hereinabove; and then hermetically sealing the single dose container. It is intended that these filled containers will allow rapid dissolution of the solid composition upon reconstitution with water in situ giving an appropriate sterile solution of desired cyclophosphamide concentration for administration. By suitable containers is meant a container capable of maintaining a sterile environment such as a vial capable of being hermetically sealed by a stopper means. Additionally, suitable containers implies appropriateness of size, considering the volume of solution to be held upon reconstitution of the lyophilized composition; and appropriateness of container material, generally Type I glass. The stopper means employed, e.g. sterile rubber closures or an equivalent, should be understood to be that which provides the aforementioned seal but which also allows entry for the purpose of introduction of diluent, e.g. sterile water, for reconstitution of the desired cyclophosphamide solution. These and other aspects of the suitability of containers for pharmaceutical products such as those of the instant invention are well known to those skilled in the practice of pharmaceutical arts.

While the physical properties, such as appearance and particularly dissolution time, were improved in the instant solid compositions, thereby achieving one objective of the invention, we unexpectedly found that these instant solid compositions also possessed improved thermal stability compared with currently known dry powder premix formulations as well as cyclophosphamide itself. In practice, expectation for enhancement of chemical stability by lyophilization relates to a comparison of the lyophilizate solid with the solution form of the pharmaceutical composition. In contrast, the instant compositions demonstrate enhanced chemical stability between solid dosage forms, cf: Table 4.

                  TABLE 4                                                          ______________________________________                                         Comparative Chemical Stabilities for "Hydrated"                                Cyclophosphamide/Mannitol Lyophilizate and                                     CYTOXAN ® for Injection (Powder Premix)                                    Storage     Time    Potency (% of Zero-time)                                   Temperature (Wks)   Lyophilizate CYTOXAN ®                                 ______________________________________                                         50°  1        31          31                                            45°  1        94          80                                                        2        76          42                                            40°  1       100          98                                                        3       101          85                                                        6        99          33                                                        12       98           2                                            35°  3       101          105                                                       6       100          98                                                        12      100          98                                                        26       98          85                                            25°  6       100          101                                                       12      100          100                                                       26      100          103                                           ______________________________________                                    

It is recognized by practitioners in the pharmaceutical arts that the use of elevated storage temperatures for shorter time periods is standard practice in obtaining comparative stability data. As can be seen in Table 4, the "hydrated" cyclophosphamide/mannitol lyophilized solid composition possesses greater thermal stability than the current CYTOXAN® for Injection dry powder formulation.

Short term comparative stability studies have also been made using the "hydrated" cyclophosphamide/mannitol lyophilized solid composition, a cyclophosphamide monohydrate/mannitol dry powder blend (not lyophilized), CYTOXAN® for Injection dry powder premix, and pure cyclophosphamide monohydrate. These studies at a storage temperature of 40° C. gave the results summarized in Table 5.

                                      TABLE 5                                      __________________________________________________________________________         Potency                                                                    Time                                                                               (% of Zero-time)                                                                          Physical Appearance                                             (Wks)                                                                              A  B C   D A     B    C    D                                               __________________________________________________________________________     3   104                                                                               96                                                                               93  95                                                                               unchanged                                                                            sl. melted                                                                          sl. melted                                                                          unchanged                                       6   102                                                                               57                                                                               39  37                                                                               unchanged                                                                            melted                                                                              melted                                                                              melted                                          12  101                                                                               42                                                                                4  --                                                                               unchanged                                                                            melted                                                                              melted                                                                              --                                              __________________________________________________________________________      Comparative Stabilities at 40° C. for:                                  A. "Hydrated" Cyclophosphamide/Mannitol Lyophilizate                           B. Cyclophosphamide Monohydrate/Mannitol Powder Blend                          C. CYTOXAN ® for Injection (Powder Premix)                                 D. Cyclophosphamide Monohydrate                                          

As these tabular results show, the "hydrated" cyclophosphamide/mannitol lyophilized solid composition demonstrates superior thermal stability as well as maintenance of initial physical appearance. In summary, the "hydrated" cyclophosphamide/mannitol lyophilized solid composition shows unexpected improved stability, superior solubility characteristics, and enhanced appearance when compared with other powder premix formulations of cyclophosphamide monohydrate and cyclophosphamide monohydrate alone. These improved characteristics are indicative of a superior pharmaceutical solid composition.

The following examples describe in detail methods for preparation of a solid composition of the present invention. It will be apparent to those skilled in the art that many modifications, both of methods and materials may be practiced without departing from the purpose and intent of this disclosure. From the foregoing description and the following examples, it is believed that one skilled in the art is able to use the invention to the fullest extent.

EXAMPLE 1 Lyophilization--Humidification Process

An aqueous solution is typically prepared by dissolving mannitol (375 g) and cyclophosphamide monohydrate (535 g) in 13 liters of Water for Injection. Additional Water for Injection is added in order to bring the final volume of this solution to 15 liters. The solution is first passed through a sterile prefilter and then a sterile 0.22 μm pore size membrane filter, following which the sterile solution is then aseptically filled into sterile 30 mL glass vials. Each vial is filled with 15.0 mL solution (containing the equivalent of 500 mg anhydrous cyclophosphamide) and then sterile rubber closures are aseptically inserted in the lyophilization position. These filled vials are placed in suitable lyophilization equipment and cooled as quickly as possible to a temperature of about -35° C.

The lyophilizer condenser is then cooled to about -60° C. and the lyophilization chamber is evacuated to a pressure of approximately 500 millitorr. Shelf heat is set at +20° C. to begin the drying process. Following about 15-20 hours of primary drying, the shelf temperature is raised in order to bring the product temperatures to near +25° C. provided that the product temperature thermocouples have reached +15° to +18° C. and the lyophilization chamber pressure is not more than 500 millitorr at that point. Lyophilization is then continued until a final product temperature of +22° to +24° C. with a chamber pressure of not more than about 200 millitorr, is reached. Depending on the capacity of the lyophilization equipment and the product batch size, the total lyophilization time will vary but is generally in the range of 24-36 hours. Following completion of the lyophilization process, the high vacuum is relieved by the aseptic introduction of sterile air and/or nitrogen having 80% relative humidity at room temperature. An aseptic environment is maintained during this rehydration process which is complete within approximately 18-24 hours at atmospheric pressure. At this point, the vials are closed by aseptically seating the lyophilization stoppers into the vials by mechanical collapse of the shelves.

A preferred method of humidification involves introducing water vapor into the lyophilization chamber while it remains under reduced pressure. With the chamber at about 25° C. and with a closed vacuum (pump off) of about 100-200 millitorr, a quantity of sterile water is vaporized into the chamber during which time the pressure may increase to about 25 torr. The quantity of sterile water used depends on the size of the lyophilization equipment being used as well as the product batch size. Typically, for 180 vials of the size and content described hereinabove and with a lyophilization chamber volume of approximately 125 liters, the amount of water used is on the order of 9 mL. The introduction of water is accomplished by establishing a closed system and bleeding in the water vapor issuing from a reservoir of liquid water kept at about 60°-80° by external heating. Boiling chips are used to prevent bumping and the introduction of water is completed within approximately 45 minutes. The internal chamber pressure during this "vacuum humidification" process varies from about 0.1 to 25 torr with the introduction of the water vapor. Total time of humidification using this method generally requires about 2-6 hours, again depending on lyophilization equipment size and product batch size. After humidification, the remaining vacuum is relieved by introduction of sterile air and/or nitrogen and the vials are closed by aseptically seating the lyophilization stoppers into the vials by mechanical collapse of the shelves.

EXAMPLE 2 Direct Lyophilization Process

Utilizing the filled vials with stoppers as described above in Example 1, the lyophilization equipment is loaded and the vials cooled as rapidly as possible to a temperature of about -35° C. For stage 1 of this freeze-drying process the lyophilizer condenser is cooled to about -60° C., the lyophilization chamber is evacuated to approximately 500 millitorr, and the shelf is held at a temperature on the order of +10° C.

When the product temperature (measured by thermocouple) reaches about -12° C., generally after about 10-12 hours, adjustments are made for the second stage of freeze-drying. The shelf temperature is lowered to about -10° C. and the condenser temperature is raised to about -30° C. Depending on capacity of the equipment being used and the size of the product batch, approximately 36-48 hours of total lyophilization time is required in order to reach equilibrium in the chamber and for the product to reach the desired final product temperature, as measured by thermocouple, of about -10° C. When this second stage of freeze-drying of the lyophilization process is complete, the vacuum is relieved by the aseptic introduction of sterile nitrogen and/or air. The containers are then closed as in Example 1 above.

The lyophilized solid compositions resulting from use of the processes outlined above in Examples 1 and 2 contain, within ±10%, 500 mg cyclophosphamide (on the anhydrous basis) and 375 mg of mannitol. While the intended amount of water is 35 mg (equimolar with 500 mg of anhydrous cyclophosphamide), in actual practice this amount varies from about -10% to about +30%.

The sealed vials described in Examples 1 and 2 are intended for use as single dose formulations following reconstitution with about 25 mL of Water for Injection. The reconstituted solutions may be administered either parenterally or orally. Additional single dose formulations are shown in Table 6. The values listed for volumes and weights are the nominal values and may be expected to vary as mentioned hereinabove.

                                      TABLE 6                                      __________________________________________________________________________     Additional Single Dose Formulations                                            Lyophilizate Compositions   Volume of Water                                    Example                                                                             Cyclophosphamide                                                                          Mannitol    for Reconstitution                                 No.  (mg, anhydrous basis)                                                                     (mg)  H.sub.2 O (mg)                                                                       (mL)                                               __________________________________________________________________________     3     100        75    7     5                                                 4     200       150   14    10                                                 5    1000       750   70    50                                                 6    2000       1500  140   100                                                __________________________________________________________________________

Further Detailed Description of the Invention

A number of additional examples of solid compositions of the instant invention similar to those defined hereinabove have been prepared. These additional examples of the lyophilized pharmaceutical solid compositions serve to extend the range of the excipient, comprised of mannitol, upward to about 85 parts by weight in the composition. Examples of the solid compositions of the instant invention disclosed hereinbefore contain an excipient range of from about 10 to 40 parts in the final composition. These compositions resulted from lyophilization of solutions containing about 3.33% W/V cyclophosphamide, on an anhydrous basis, and having mannitol concentrations of from 1.7 to 7.0% W/V. Lyophilization of these solutions (cf: Table 1, page 9) resulted in good and acceptable lyophilizate cakes as evaluated according to the criteria of physical appearance (original shape, no shrinkage or meltback, good coloration, homogeneity, firmness, and crystallinity) and an acceptable dissolution rate upon reconstitution. Results obtained by the lyophilization of solutions containing 3.33% W/V cyclophosphamide but with mannitol concentrations of 10 or 15% produced poor or unacceptable lyophilizate cakes when judged by the above criteria. These cakes were not homogeneous, did not retain their original shape, and/or did not dissolve rapidly upon reconstitution.

Continuing study was directed to determine the upper boundary of the range of the mannitol excipient which could be achieved in an acceptable solid composition of the instant invention. Production of these high mannitol content solid compositions requires some alteration of the processes as previously described, particularly in regard to the initial pre-lyophilization solution employing a lower cyclophosphamide concentration and progressively larger amounts of water. Examples of these higher mannitol excipient content compositions are shown below in Table 7.

                                      TABLE 7                                      __________________________________________________________________________                          Solution Fill                                             Pre-lyophilization   Volume (mL) for                                                                          Lyophilizate                                    Solution Concentration (% W/V)                                                                      500 mg    Composition                                     Cyclophosphamide (CY)                                                                      Mannitol (MAN)                                                                          Cyclophosphamide                                                                         (parts by weight)                               __________________________________________________________________________     3.33        7.00     15.00     CY 20, MAN 42,                                                                 H.sub.2 O 1.4                                   2.67        7.00     18.75     CY 20, MAN 52.5,                                                               H.sub.2 O 1.4                                   2.22        7.00     22.50     CY 20, MAN 63,                                                                 H.sub.2 O 1.4                                   1.90        7.00     26.2      CY 20, MAN 73.5,                                                               H.sub.2 O 1.4                                   1.67        7.00     30.00     CY 20, MAN 84,                                                                 H.sub.2 O 1.4                                   __________________________________________________________________________

To summarize; the solid compositions of the present application have the following ingredients and concentrations:

cyclophosphamide--about 19 to 64% by weight,

water--about 1 to 5% by weight,

mannitol--about 32 to 80% by weight,

preferred compositions:

cyclophosphamide--about 32 to 64% by weight,

water--about 2 to 5% by weight,

mannitol--about 32 to 66% by weight; and

the most preferred composition:

cyclophosphamide--about 55% by weight,

water--about 4% by weight,

mannitol--about 41% by weight.

All of the above solid compositions possess the characteristics required for the improved solid compositions of the present invention. However, since lowering the cyclophosphamide concentration in the pre-lyophilization solution, in effect, requires the removal of more water during lyophilization, the process requires more time and is therefore, less efficient and more expensive. Thus, while lowering the cyclophosphamide concentration in the pre-lyophilization solution, containing mannitol at the 7.0% W/V level, has no detrimental effect on the quality of the final compositions, the decreased efficiencies in their production render them less preferred, though still operable, than the original compositions containing a final mannitol content of from about 10 to about 40 parts by weight. 

What is claimed is:
 1. A process for producing a lyophilized cyclophosphamide pharmaceutical solid composition with improved stability, superior solubility, and enhanced appearance which comprises(a) dissolving about two parts by weight of cyclophosphamide monohydrate and from about 1-8 parts by weight of excipient comprising mannitol in sufficient water to give about a 1.67 to 4% (W/V) concentrated cyclophosphamide solution; (b) filling a container with said solution to a desired cyclophosphamide content; (c) lyophilizing said solution of desired cyclophosphamide content in said container to a dryness of about 2% or less; and (d) humidifying the lyophilizate by means of exposure to water vapor at about room temperature and a pressure of from about 100 millitorr to atmospheric pressure for an effective time of at least 2 hours until about 1 equivalent of water per equivalent of anhydrous cyclophosphamide is taken up.
 2. The process of claim 1 wherein lyophilization step (c) comprises rapidly freezing said solution of desired cyclophosphamide content and then employing a chamber pressure of about 50-500 millitorr, a shelf temperature of about 20°-25°, and a condenser temperature of about -60° for about 18-36 hours in order to reduce the moisture in the container to about 2% or less.
 3. The process of claim 1 wherein the rehydration step (d) comprises exposing the lyophilizate to air with a controlled humidity of about 60-80% at atmospheric pressure and room temperature for about 1-2 days.
 4. The process of claim 1 wherein the rehydration step (d) comprises relieving the vacuum of the lyophilization chamber with a gas which can be air or nitrogen containing about 80% relative humidity at room temperature and holding the lyophilizate in contact with this moist atmosphere for about 18-24 hours until about one equivalent of water per equivalent of anhydrous cyclophosphamide is taken up.
 5. The process of claim 1 wherein the rehydration step (d) is accomplished by exposing the lyophilizate to water vapor at a pressure of about 100 millitorr to 25 torr and about 25° for about 2-6 hours until about 1 equivalent of water per equivalent of anhydrous cyclophosphamide is taken up.
 6. The process of claim 1 wherein the rehydration step (d) is accomplished by exposing the lyophilizate to water vapor at a pressure of about 100 millitorr to 25 torr and about 25° for about 2-6 hours until about 1 equivalent of water per equivalent of anhydrous cyclophosphamide is taken up.
 7. A process for producing lyophilized cyclophosphamide pharmaceutical solid composition with improved stability, superior solubility, and enhanced appearance which comprises(a) dissolving about 2 parts by weight of cyclophosphamide monohydrate and from about 1-8 parts by weight of excipient comprising mannitol in sufficient water to give about a 1.67 to 4% (W/V) concentrated cyclophosphamide solution; (b) filling a container with said solution to a desired cyclophosphamide content; (c) lyophilizing said solution at a chamber pressure of about 500 millitorr and an initial shelf temperature of about 10° and an initial lyophilization condenser temperature of about -60° for about 10-12 hours; (d) continuing the lyophilization of said solution at a chamber pressure of about 500 millitorr but with a shelf temperature of about -10° and a condenser temperature of about -30° for a total lyophilization time of 36-48 hours at which point the amount of water remaining is approximately equimolar with the cyclophosphamide on an anhydrous basis.
 8. A process for producing a lyophilized cyclophosphamide pharmaceutical solid composition with improved stability, superior solubility, and enhanced appearance which comprises(a) dissolving from about 2 parts by weight of cyclophosphamide monohydrate and from about 1-8 parts by weight of excipient comprising mannitol in sufficient sterile water to give about a 1.67 to 4% (W/V) concentrated cyclophosphamide solution; (b) filtering the cyclophosphamide solution and then filling a container under sterile conditions, with the filtered cyclophosphamide solution to a desired cyclophosphamide content; (c) lyophilizing the solution of desired cyclophosphamide content in the container to a dryness of about 2% or less moisture by rapidly freezing the solution in its container and then subjecting the frozen cyclophosphamide solution to lyophilization conditions comprising a chamber pressure of about 50-500 millitorr, a condenser temperature of about -60°, and a shelf temperature of about 0°-25°, for about 14-36 hours; (d) humidifying the lyophilizate by exposing it to water vapor at a pressure of about 100 millitorr to 25 torr at about 25° for about 2-6 hours until about 1 equivalent of water per equivalent of anhydrous cyclophosphamide is taken up; and (e) aseptically sealing the container which contains the lyophilized cyclophosphamide pharmaceutical solid composition.
 9. A process for producing a lyophilized cyclophosphamide pharmaceutical solid composition with improved stability, superior solubility, and enhanced appearance which comprises(a) dissolving from about 2 parts by weight of cyclophosphamide monohydrate and from about 1-8 parts by weight of excipient comprising mannitol in sufficient sterile water to give about a 1.67 to 4% (W/V) concentrated cyclophosphamide solution; (b) filtering the cyclophosphamide solution and then filling a container under sterile conditions, with the filtered cyclophosphamide solution to a desired cyclophosphamide content; (c) initially lyophilizing the solution of desired cyclophosphamide content in the container by rapidly freezing the solution in its container and then subjecting the frozen cyclophosphamide solution to lyophilization conditions comprising a chamber pressure of about 500 millitorr, an initial shelf temperature of about 10°, and an initial lyophilization condenser temperature of about -60° for about 10-12 hours; (d) continuing the lyophilization of the frozen solution at a chamber pressure of about 500 millitorr but with a shelf temperature of about -10° and a condenser temperature of about -30° for a total lyophilization time of 36-48 hours at which point the amount of water remaining is approximately equimolar with the cyclophosphamide on an anhydride basis; and (e) aseptically sealing the container which contains the lyophilized cyclophosphamide pharmaceutical solid composition.
 10. In a process for freeze-drying a dosage amount of cyclophosphamide hydrate from an aqueous solution the improvement comprising carrying out said process in two stages, the first stage comprising freeze-drying a solution of cyclophosphamide hydrate in combination with from about 0.5 times to about 2 times its weight of an excipient in which mannitol is present as the major excipient by weight, until the moisture content of freeze-dried material is less than 2% by weight of the freeze-dried material, and the second stage comprising rehydrating the freeze-dried material by contacting it with water vapor until the relative humidity to which the material is exposed is about 60-80% and maintaining the relative humidity at this level so that the moisture content of the product is in the range from about 2% to about 7% based on the total net weight of product, and the integrity of said cyclophosphamide hydrate is maintained. 